The present invention relates to siRNA specific to mortalin and to the use of same for decreasing the levels of mortalin for disease treatment.
Diseases such as tumoral, infectious, autoimmune and transplantation-related diseases, which are associated with pathological cells and are treatable via complement-mediated cytolysis of such cells, represent numerous highly debilitating and/or lethal diseases for which no optimal therapy exists. There is therefore a long-felt and urgent need in the art for novel and maximally effective methods and therapeutic agents for treating such diseases.
The complement system consists of more than twenty blood plasma proteins that cooperate with other sections of the innate and acquired immune systems in clearance of pathogenic organisms, immune complexes and apoptotic cells. The complement activation cascade culminates in formation of the membrane attack complex (MAC), made of complement C5b, C6, C7, C8 and C9 proteins (termed “C5b-9”), and its insertion into the plasma membrane of target cells. Membrane insertion begins when C5b-7 forms, is enhanced upon formation of C5b-8 complex and is maximal upon binding and oligomerization of C9 and formation of a transmembrane, cylinder-shape polyC9 complex attached to C5b-8. At supralytic doses, MAC normally functions to induce rapid cell death by necrosis (Koski, C. L. et aL, 1983. Proc Natl Acad Sci USA 80:3816) or apoptosis (Cragg, M. S. et al., 2000. Cell Death Differ 7:48). At low, sublytic doses, MAC acts as a potent stimulator of numerous cellular activities (for review see Bohana-Kashtan, O. et al., 2004. Mol Immunol 41:583). Treatment with sublytic MAC has been shown to transduce either anti-necrotic (Reiter, Y. et al., 1992. Eur J Immunol 22:1207) or anti-apoptotic (Dashiell, S. M. et al., 2000. Glia 30:187) signals into various cells.
As a means of protection from complement, nucleated cells can remove the MAC from their plasma membrane by endocytosis, vesiculation or proteolytic fragmentation. Physical removal of MAC by vesiculation has been demonstrated in several cell types including neutrophils, oligodendrocytes and platelets, and in the tumor cell lines U937 and K562 (Sims, P. J. and Wiedmer, T. 1986. Blood 68:556; Scolding, N. J. et al., 1989. Nature 339:620; Morgan, B. P. et al., 1986. J Immunol 136:3402; Morgan, B. P. 1992. Curr Top Microbiol Immunol 178:115). To date, little is known about the molecular mechanism responsible for MAC vesiculation. Yet, removal of complement from nucleated cells may be associated with disease pathogenesis. For example, MAC removal has been shown to protect cancer cells from complement-mediated cytotoxicity (Carney D. F., J Immunol 134:1804, 1985; Pilzer D. and Fishelson Z., Int Immunol 17:1239, 2005). Furthermore, membrane complement regulatory proteins (mCRPs) have been shown to be over-expressed on the surface of cancer cells and render them resistant to autologous complement (Fishelson Z. et al., 2003. Mol Immunol 40:109-23).
Mechanisms protecting cells from heat-shock and from complement share some resemblance. For example, both of these shock responses depend on de-novo protein synthesis, exhibit similar functional kinetics, and studies have suggested a role for members of the 70 kilodalton heat shock protein (HSP70) family proteins in regulation of complement-mediated cytolysis (Fishelson Z. et al., 2001. Int Immunol. 13:983-991).
Mortalin, also known as GRP75, PBP74, mitochondrial HSP75 and mot-2, is a member of the HSP70 family (GeneCard #GC05M137967). This protein has been assigned multiple functions including stress response (Carette, J. et al., 2002. Int J Radiat Biol 78:183), glucose regulation, p53 inactivation, control of cell proliferation, differentiation, tumorigenesis and mitochondrial import (reviewed in Wadhwa, R. et al., 2002. Cell Stress Chaperones 7:309; Voisine, C. et al., 1999. Cell 97:565). Mortalin is thought to act as an intracellular protein, in mitochondria and several other cytoplasmic locations such as endoplasmic reticulum and cytoplasmic vesicles (Ran, Q. et al., 2000. Biochem Biophys Res Commun 275:174). Mortalin is ubiquitously and constitutively expressed in normal tissues, and has been shown to be displayed on the surface of mouse B-cells and macrophages (VanBuskirk, A. M. et al., 1991. J Immunol 146:500). Its expression level is upregulated in some tumors, such as neuroblastoma, lung adenocarcinoma, leukemia and ovarian cancer cells (Takano, S. et al., 1997. Exp Cell Res 237:38; Dundas, S R. et al., 2004. J Pathol 205:74; Shin, B. K. et al., 2003. J Biol Chem 278:7607), as well as during infection and inflammation (Kirmanoglou, K. et al., 2004. Basic Res Cardiol 99:404; Johannesen, J. et al., 2004. Autoimmunity 37:423). Overexpression of mortalin in normal cells considerably extends their lifespan (Kaul, S. C. et al., 2003. Exp Cell Res 286:96), while reduction of mortalin levels in immortalized cells causes growth arrest (Wadhwa, R. et al., 2004. J Gene Med 6:439; Wadhwa et al., 1994. Cell Struct Funct 19:1-10). In view of the expression of mortalin in cancers, the use of this protein as therapeutic target has been proposed (Wadhwa R. et al., 2002. Histol Histopathol 17:1173-7).
Several approaches have been proposed involving decreasing the levels/activity of HSP70 family proteins, such as mortalin, for treating diseases associated with pathological cells and treatable via complement-mediated cytolysis of such cells.
One approach involves administration of the mortalin inhibitor MKT-077 (formerly FJ-776) for treatment of cancers characterized by wild-type p53 (Wadhwa R. et al., 2000. Cancer Research 60, 6818-6821), chemo-resistant solid tumors (Propper D. J. et al., 1999. Ann. Oncol., 10: 923-927), untreatable/treatment-refractory solid tumors (Britten C. D. et al., 2000. Clin Cancer Res., 6: 42-49), or solid tumors of various lineages (Wadhwa R. et al., 2002. Cancer Res. 62:4434-8).
Yet another approach involves targeting mortalin using conventional and RNA-helicase-coupled hammerhead ribozymes for the treatment of cancers (Wadhwa R. et al., 2003. EMBO Rep 4: 595-601).
An additional approach suggests using mortalin as molecular target for treatment of hepatitis C virus-related hepatocellular carcinoma (Takashima M. et al., 2003. Proteomics. 3: 2487-93).
A further approach suggests employing inhibition of HSC70 with deoxyspergualin to increase the sensitivity of K562 human erythroleukemia cells to complement-mediated lysis (Fishelson Z. et al., 2001. Int Immunol. 13: 983-991).
Yet a further approach involves expression of mortalin anti-sense RNA in cancer cells for treatment of cancers characterized by compromised p53 and pRB functions and telomerase activity (Wadhwa R. et al., 2004. J Gene Med. 6: 439-44).
U.S. Publication No. 20060270622 discloses means of treating diseases associated with pathological cells by modulating the levels of mortalin in these cells and thus effecting the association of these cells with the complement system. According to the teachings of U.S. Publication No. 20060270622, decreasing the levels of mortalin level/activity can be affected by the use of anti-mortalin antibodies or by transfection with siRNA specific to mortalin. However, specific sequences of siRNA capable of down-regulating mortalin were not disclosed.
There is thus a widely recognized need for, and it would be highly advantageous to have maximally effective siRNAs capable of decreasing the levels of mortalin for the treatment of diseases such as cancer.